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10.1016/j.ab.2015.08.025 http://scihub22266oqcxt.onion/10.1016/j.ab.2015.08.025 C4615133!4615133 !26334609     free     free     free Warning: file_get_contents(https://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=26334609 &cmd=llinks): Failed to open stream: HTTP request failed! 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Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase |pdf=|usr=}}{{26334609 }} gab.com Text Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase JO: Anal Biochem http://www.kidney.de/pget.php?&tw=1&pmid=26334609 #FOAvip Hedgehog signaling is critical for correct embryogenesis and tissue development. However, on maturation, signaling is also found to be aberrantly activated in many cancers. Palmitoylation of the secreted signaling protein sonic hedgehog (Shh) by the enzyme hedgehog acyltransferase (Hhat) is required for functional signaling. To quantify this important posttranslational modification, many in vitro Shh palmitoylation assays employ radiolabeled fatty acids, which have limitations in terms of cost and safety. Here we present a click chemistry armed enzyme-linked immunosorbent assay (click-ELISA) for assessment of Hhat activity through acylation of biotinylated Shh peptide with an alkyne-tagged palmitoyl-CoA (coenzyme A) analogue. Click chemistry functionalization of the alkyne tag with azido-FLAG peptide allows analysis through an ELISA protocol and colorimetric readout. This assay format identified the detergent n-dodecyl ?-d-maltopyranoside as an improved solubilizing agent for Hhat activity. Quantification of the potency of RU-SKI small molecule Hhat inhibitors by click-ELISA indicated IC50 values in the low- or sub-micromolar range. A stopped assay format was also employed that allows measurement of Hhat kinetic parameters where saturating substrate concentrations exceed the binding capacity of the streptavidin-coated plate. Therefore, click-ELISA represents a nonradioactive method for assessing protein palmitoylation in vitro that is readily expandable to other classes of protein lipidation. Twit Text FOAVip Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase ????Anal Biochem http://www.kidney.de/pget.php?&tw=1&pmid=26334609 #FOAvip Twit Text # Free Paper on # # # # # LINK http://www.kidney.de/pget.php?&tw=1&pmid=26334609 #FOAvip English Wikipedia <ref>{{Cite pmid|26334609 }}</ref> Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase #MMPMID26334609 Lanyon-Hogg T ; Masumoto N ; Bodakh G ; Konitsiotis AD ; Thinon E ; Rodgers UR ; Owens RJ ; Magee AI ; Tate EW Anal Biochem 2015[Dec]; 490 (ä): 66-72 PMID26334609 show ga Hedgehog signaling is critical for correct embryogenesis and tissue development. However, on maturation, signaling is also found to be aberrantly activated in many cancers. Palmitoylation of the secreted signaling protein sonic hedgehog (Shh) by the enzyme hedgehog acyltransferase (Hhat) is required for functional signaling. To quantify this important posttranslational modification, many in vitro Shh palmitoylation assays employ radiolabeled fatty acids, which have limitations in terms of cost and safety. Here we present a click chemistry armed enzyme-linked immunosorbent assay (click-ELISA) for assessment of Hhat activity through acylation of biotinylated Shh peptide with an alkyne-tagged palmitoyl-CoA (coenzyme A) analogue. Click chemistry functionalization of the alkyne tag with azido-FLAG peptide allows analysis through an ELISA protocol and colorimetric readout. This assay format identified the detergent n-dodecyl ?-d-maltopyranoside as an improved solubilizing agent for Hhat activity. Quantification of the potency of RU-SKI small molecule Hhat inhibitors by click-ELISA indicated IC50 values in the low- or sub-micromolar range. A stopped assay format was also employed that allows measurement of Hhat kinetic parameters where saturating substrate concentrations exceed the binding capacity of the streptavidin-coated plate. Therefore, click-ELISA represents a nonradioactive method for assessing protein palmitoylation in vitro that is readily expandable to other classes of protein lipidation. |*Protein Processing, Post-Translational/drug effects [MESH] |Acyltransferases/antagonists & inhibitors/chemistry/genetics/*metabolism [MESH] |Biotinylation [MESH] |Click Chemistry [MESH] |Detergents/chemistry [MESH] |Enzyme Inhibitors/pharmacology [MESH] |Enzyme-Linked Immunosorbent Assay [MESH] |Fatty Acids, Unsaturated/pharmacology [MESH] |HEK293 Cells [MESH] |Hedgehog Proteins/chemistry/*metabolism [MESH] |Humans [MESH] |Immobilized Proteins/chemistry/metabolism [MESH] |Lipoylation/drug effects [MESH] |Maltose/analogs & derivatives/chemistry [MESH] |Oligopeptides/chemistry/metabolism [MESH] |Palmitoyl Coenzyme A/analogs & derivatives/metabolism [MESH] |Peptide Fragments/chemistry/metabolism [MESH] |Recombinant Fusion Proteins/chemistry/metabolism [MESH] |Solubility [MESH] |Streptavidin/chemistry/metabolism [MESH]Click chemistry armed enzyme-linked immunosorbent assay to measure pal(epmc).pdf DeepDyve Pubget Overpricing